Open AccessDetection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears
Author(s) -
Mazhgan Rowneki,
Naomi Aronson,
Peicheng Du,
Paige Sachs,
Robert J. Blakemore,
Soumitesh Chakravorty,
Shawn Levy,
Angela L. Jones,
Geetika Trivedi,
Sheilla Chebore,
Dennis Addo,
Denis K. Byarugaba,
Panganani Njobvu,
Frederick Wabwire-Mangen,
Bernard Erima,
Eric Ramos,
Carlton A. Evans,
Braden Hale,
James D. Mancuso,
David Alland
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0232343
Subject(s) - rpob , drug resistance , mycobacterium tuberculosis , tuberculosis , inha , dna extraction , microbiology and biotechnology , biology , medicine , polymerase chain reaction , bacilli , virology , gene , bacteria , pathology , genetics
Background Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs. Methods We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB , katG , embB , gyrA , gyrB , rpsL , and rrs genes, the inhA , eis , and pncA promoters and the entire pncA gene. Results We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases. Conclusion This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.